It has been appreciated for some time that isotopic analysis of compounds provides a wealth of information extending beyond traditional structural chemical analyses. While quantitative and qualitative structural analyses identify the chemical composition of a compound or of individual molecules, isotopic analysis provides still further information concerning the source, origin and formation of such compounds and molecules.
In the field of geochemical oil exploration and prospecting, measurement of the isotopic compositions of large numbers of individual organic compounds of oil samples from various oil reservoirs assists in clarifying the origins of specific compounds, correlating the organic compounds with particular sources, recognizing the existence of multiple sources, examining the mechanisms of petroleum generation and improving the sensitivity of petroleum migration studies. This information, particularly in connection with seismic data, can be used to predict locations of other oil reservoirs to which oil may have migrated from a common source of generation or formation.
Isotope ratio monitoring has further applications in the biomedical field, wherein non-radioactive, stable isotopes are used as tracer labels in drug metabolism and other biomedical studies and where natural variations in isotopic abundances may also carry additional information regarding sources and fates of metabolites. Current non-radioactive, stable isotopic labelling apparatus and methods in the medical field employ costly labelled compounds having isotope ratios much greater than those found in natural abundance. Improvements in isotope ratio monitoring sensitivity and precision and a reduction in the size of required samples and in the time and complexity of isotopic analyses, however, will enhance applications in the biomedical field for non-radioactive, stable isotope ratio monitoring apparatus and methods.
Additional applications lie within the environmental sciences. Even though a pollutant may have several potential sources, it may happen that its isotopic composition will match that of only one of them, thus clearly identifying the source. In other cases, it may happen that a pollutant has an isotopic composition intermediate between those of two possible sources, thus allowing determination of the importance of each source.
Isotopic analysis is used to determine the relative abundance of various isotopes in a sample compound. In analyses of carbon isotopes, for example, the ratio of .sup.1 3 C to .sup.1 2 C in a sample compound is determined. Such ratio is typically compared or normalized with that of a known compound or standard. Several prior art methods and apparatus have been used in an effort to automate isotopic analysis of sample compounds. Each such method suffers from at least one of several deficiencies, which include, among other things, the labor intensity and substantial time required to perform the analysis, a lack of precision in the results, and requirements for large volumes of sample to perform the analysis.
In a first category of prior art methods and apparatus, commonly referred to as selected ion monitoring gas chromatography-mass spectrometry (SIM-GCMS), a gas chromatograph is directly connected to a single collector mass spectrometer of the type typically used for organic analysis (e.g., a magnetic sector or quadrupole mass spectrometer). The compound to be analysed is purified by elution from the gas chromatograph (or by other conventional means) and is introduced as intact molecules into the mass spectrometer ion source. The mass spectrometer fragments the compound in accordance with a known and expected pattern and the single detector is employed to ascertain the relative abundances of fragment ions at varying masses. Isotopic abundances of the sample compound are distinguishable from the mass spectrometer data based upon the differences in ion current intensities at the selected masses.
The SIM-GCMS apparatus and methods advantageously require only relatively small sample amount (picomoles to nanomoles). Moreover, because the entire analysis is done in a continuous, uninterrupted, automated sequence (i.e., "on-line"), the procedure is relatively uncomplicated, requiring minimal time and human intervention.
The above-noted advantages, however, are achieved at the expense of precision. Specifically, since the ion source of the mass spectrometer must be arranged so that thermal degradation of organic molecules is minimized, molecular residence times are brief and, as a result, efficiencies of ionization are often low. Ion currents are commonly in the range 10.sup.-1 1 to 10.sup.-1 5 amperes, and theoretical maximum precisions of ratio measurement (within the time of a gas chromatographic peak) are often only a few percent. Further, since a single detector is used to monitor the ion currents both .sup.1 2 C and .sup.1 3 C isotopic abundances, it must have a linear response for the range of abundances of both. Scanning with a single detector during the course of a chromatographic peak introduces systematic errors into the isotopic ratio determination. Although attempts been made to correct for such errors by altering the scan pattern (e.g., by adopting a unidirectional scan pattern), all such corrections are based upon the uncertain assumption that no fractionation occurs across the chromatographic peak for the eluted composition, i.e., that the compound is homogenous throughout the chromatographic peak.
Also, precision losses in SIM-GCMS apparatus and methods stem from the introduction of relatively large and complex molecules into the mass spectrometer directly as they are eluted from the gas chromatograph. The size of these molecules demands higher resolution from the mass spectrometer. The inevitable presence of the element hydrogen creates a situation in which loss or transfer (i.e., between molecular fragments) of hydrogen can produce a mass change of only one mass unit, exactly that associated with the presence or absence of a single carbon- or nitrogen-isotopic label. For example, an ion thought to contain an isotopic label may instead have picked up an extra hydrogen atom during fragmentation. An ion that has unexpectedly lost a hydrogen atom may be mistakenly identified as one that does not contain an isotopic label. Furthermore, as noted above, the molecules fragment in the ion source of the mass spectrometer which decreases the intensity of parent ions and complicates isotopic analysis and corrections for abundances of non-carbon isotopes (e.g., .sup.2 H and .sup.1 7 O). Furthermore, the extent of fractionation between isotopes of the parent ion in the fragmentation process is unknown. It is thus not known whether one isotope will tend to fragment from the parent ion more often than another isotope, introducing still further possible errors in the measurement of the relative ratios of isotopes.
Because of the poor precision of SIM-GCMS techniques (e.g., 0.5 to 10.0%, depending upon conditions), their application to isotope monitoring have generally been limited to the detection of artificially labelled compounds having .sup.1 3 C abundances substantially greater than the natural .sup.1 3 C abundance in organic compounds.
The precision limitations of SIM-GCMS techniques led to isotopic analysis methods in which compounds are quantitatively combusted after being resolved by the gas chromatograph and before introduction into the mass spectrometer. Such combustion converts the carbon in the larger organic molecules to .sup.1 2 CO.sub.2 and .sup.1 3 CO.sub.2, which can then be analyzed with far greater efficiency. Because CO.sub.2 is a small molecule containing only carbon and oxygen, fragmentation is minimized, problems of hydrogen transfer and loss are nonexistent, and calculation of carbon isotopic abundances from observed ion currents can be accomplished with far less uncertainty. Two general prior art approaches involving combustion of the effluent from a gas chromatograph before introduction into a mass spectrometer have been employed.
In a first such prior art approach, commonly referred to as the "off-line" approach, compounds are chromatographically separated and isolated, and thereafter separately analysed in a mass spectrometer. In one category of off-line apparatus and methods, the compounds eluted from the chromatographic column are individually collected and, thereafter, in a separate step, converted to CO.sub.2 by combustion, e.g., in a quartz container. Isotopic analysis is thereafter performed on the collected combustion product, which includes both .sup.1 2 CO.sub.2 and .sup.1 3 CO.sub.2.
In another category of off-line apparatus and methods, the chromatographic effluent is combusted immediately upon exit from the gas chromatograph, and the products of combustion, containing .sup.1 2 CO.sub.2 and .sup.1 3 CO.sub.2, are collected, purified, and packaged in individual containers for further analysis.
In both categories of off-line apparatus and methods, the samples are individually transferred to a mass spectrometer (preferably a dual inlet, dual (or triple) collector isotope-ratio-monitoring mass spectrometer) and analysed in a conventional manner. These apparatus and methods yield very good precision (better than 0.1% relative standard deviation). However, because sample purification and sample combustion are performed off-line, the process is labor intensive and time consuming. Moreover, large quantities of sample (of the order of micromoles) are required.
In an attempt to overcome the disadvantages inherent in off-line isotopic analysis apparatus and methods, on-line approaches have been attempted, wherein the effluent from a gas chromatograph is introduced directly into a combustion chamber, and the combustion product is then directed to a mass spectrometer in a continuous, uninterrupted, automated process. Several problems have heretofore been encountered in the direct interfacing of a gas chromatograph and mass spectrometer through a combustion interface, which problems have degraded the precision heretofore obtainable by prior art on-line apparatus and methods.
In order to maximize precision of ion-current-ratio measurement in a mass spectrometer it is necessary that the vacuum in the mass spectrometer ion source and analyzer not be degraded. Degradation of mass spectrometer vacuum reduces ion source efficiency by causing a build-up of space charge in the ionization chamber, degrading focussing and extraction of the ion beam. Further deterioration of instrument performance results from interactions between neutral molecules and ions which cause increased scattering of the ion beam in the analyzer.
Large and varying flowrates exiting from conventional gas chromatographs, however, result in excessive pressures and pressure variations at the ion source of the mass spectrometer in on-line systems. Prior art apparatus and methods have attempted to reduce ion source pressure and pressure variations by splitting the effluent from the gas chromatograph. Apparatus such as jet separators or open splits have been employed to divert carrier gas away from the mass spectrometer. With such apparatus, however, a portion of the combusted sample is also diverted from the mass spectrometer. Precision is thus degraded in two ways: (i) less material is transmitted for ionization, resulting in weaker ion beams that are more difficult to measure, and (ii) because a portion of it is removed along with the carrier gas, CO.sub.2 derived from the sample is liable to isotopic fractionation as it passes through the separator or splitter. There is, therefore, no assurance that the isotopic composition of CO.sub.2 directed to the mass spectrometer is unaltered by the diversion of CO.sub.2 by the jet separator or open split apparatus.
A palladium membrane has been used to selectively remove a hydrogen gas carrier to enrich the effluent introduced into a mass spectrometer in gas chromatography mass spectrometry (GC-MS) systems used for conventional organic analyses. Simmonds, P.G., Shoemake, G.R., Lovelock, J.E. (1970), "Palladium-Hydrogen System", Anal-Chem 42, 881-885; Lovelock, J.E., Simmonds, P.G., Shoemake, G.R. (1970). "The Palladium Generator-Separator-A Combined Electrolytic Source and Sink for Hydrogen in Closed Circuit Gas Chromatography", Anal. Chem. 42, 969-973; Dencker, W.D., Rushneck, D.R., Shoemake, G.R. (1972), "Electrochemical Cell as a Gas Chromatograph-Mass Spectrometer Interface", Anal. Chem. 44, 1753-1758. Specifically, the hydrogen carrier gas was removed by selective permeation through a palladium membrane, without any decrease in the amount of sample delivered to the mass spectrometer. Electrochemical palladium separators were used, for example, in the Viking lander spacecraft for organic GC-MS analyses on the surface of Mars The palladium separator thus makes possible the removal of a carrier gas, without loss of sample. Palladium membrane devices were largely abandoned as separators in the prior art, however, because they tended to degrade or hydrogenate samples, it was difficult to control their temperatures (the exothermic combustion of the separated hydrogen adjacent the membrane can produce a great deal of heat), and they were difficult and expensive to fabricate.
Palladium separators have not heretofore been used in connection with isotope-ratio-monitoring apparatus. In addition to the above-noted disadvantages encountered in structural GC-MS applications, prior art palladium separators were unable to accommodate the flowrates typically required in early isotope-ratio-monitoring gas chromatography - mass spectrometry (irm-GCMS) systems.
Thus, prior to the present invention, prior art attempts at automated isotope ratio monitoring have failed to provide the required quantitative precision, except with the use of time consuming, labor intensive off-line apparatus and methods requiring large amounts of sample.